[CANCER RESEARCH 62, 5158 –5167, September 15, 2002] GADD153 and 12-Lipoxygenase Mediate Fenretinide-induced Apoptosis of Neuroblastoma 1 Penny E. Lovat, Serafina Oliverio, Marco Ranalli, Marco Corazzari, Carlo Rodolfo, Francesca Bernassola, Karen Aughton, Mauro Maccarrone, Quentin D. Campbell Hewson, Andy D. J. Pearson, Gerry Melino, Mauro Piacentini, and Christopher P. F. Redfern 2 Departments of Child Health [P. E. L., Q. D. C. H., A. D. J. P., C. P. F. R.] and Endocrinology [K. A., C. P. F. R], University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom, and Department of Biology [S. O., C. R., M. P.] and IDI-Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Laboratory, Department of Experimental Medicine [M. C., M. R., F. B., M. M., G. M.], University of Rome Tor Vergata, Rome 00133, Italy ABSTRACT The synthetic retinoid fenretinide induces apoptosis of neuroblastoma cells and in vitro acts synergistically with chemotherapeutic drugs used to treat neuroblastoma. The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving cellular signaling pathways as yet incompletely defined but, in part, involving the generation of reactive oxygen species (ROS). In an attempt to characterize the mechanism of action of fenretinide, cDNA array filters were screened to identify apop- totic genes regulated in response to treatment of SH-SY5Y cells with fenretinide. Expression of the stress-induced transcription factor, GADD153, was up-regulated at both the protein and mRNA levels in response to fenretinide. Overexpression of GADD153 increased apoptosis in the presence and absence of fenretinide, whereas reduced expression of GADD153 by expression of antisense DNA abrogated the response to fenretinide. Although fenretinide is a partial retinoic acid receptor (RAR)- / agonist, RAR/ antagonists did not block the induction of GADD153 by fenretinide; conversely, the induction of GADD153 was blocked by antioxidants. Enzyme inhibitors were used to identify pathways mediating the ROS-dependent effects of fenretinide: inhibitors of phospholipase A 2 and lypoxygenases (LOX), and specific inhibitors of 12-LOX, but not 5-LOX or 15-LOX, inhibited the induction of ROS, apoptosis, and GADD153 in response to fenretinide. The inhibition of ROS and apoptosis was reversed by the addition of the 12-LOX products, 12 (S)-hydroper- oxyeicosatetraenoic acid (12-HpETE) and 12 (S)-hydroxyeicosatetraenoic acid (12-HETE). Fenretinide did not increase free arachidonic acid levels, but increased LOX activity without a detectable increase in 12-LOX protein. These results suggest that fenretinide induces apoptosis via RAR- dependent and -independent pathways in which the RAR-independent pathway is characterized by a fenretinide-dependent increase in 12-LOX activity, leading to the induction of GADD153. The targeting of 12-LOX and/or GADD153 in neuroblastoma cells may thus present a novel path- way for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity. INTRODUCTION Neuroblastoma is a common extra-cranial tumor of childhood, responsible for 15% of all pediatric deaths from malignancy. Al- though an aggressive tumor, biologically most tumors show some form of differentiation that in a small group of patients can result in spontaneous regression (1). Retinoic acid has long been known to induce differentiation of neuroblastoma in vitro, and the observation that 13-cis retinoic acid increases event-free survival when used to treat children with residual disease after chemotherapy and bone marrow transplantation (2) has led to the inclusion of retinoids in most treatment regimes. However, it has been reported that retinoic acid- induced differentiation of neuroblastoma cells may render them re- sistant to chemotherapy (3). Synthetic derivatives of retinoic acid such as fenretinide are more effective than 13-cis retinoic acid in that fenretinide is able to directly induce apoptosis of neuroblastoma in vitro (4 – 6), and this may overcome the problem of retinoic acid- induced differentiation increasing the resistance to chemotherapeutic drugs. In addition, the fact that fenretinide is synergistic with cisplatin, etoposide, or carboplatin (7) in the induction of apoptosis of neuro- blastoma cells suggests that this retinoid may be a valuable adjunct to retinoid therapy for neuroblastoma. The mechanisms of fenretinide-induced cell death of neuroblas- toma cells are complex and probably involve several overlapping pathways. Fenretinide-induced apoptosis of neuroblastoma cells has been suggested to involve RARs 3 (6). However, oxidative stress via the induction of ROS is also involved in mediating the fenretinide- induced apoptosis of neuroblastoma (7, 8) and prostate cancer cells (9, 10). Induction of apoptosis of other cancer cells by chemotherapeutic drugs results from DNA damage leading to cell death, probably as a result of increased p53 activity (11, 12), and, therefore, the synergistic induction of apoptosis between cisplatin, etoposide, and carboplatin and fenretinide in the induction of apoptosis of neuroblastoma cells may result from the activation of different pathways of cell death (7). In neuroblastoma cells, the inhibition of apoptosis by RAR antag- onists and antioxidants suggests that signaling pathways involving RARs and ROS are both required for fenretinide-induced apoptosis (6). Recent studies have also suggested that a p53-independent path- way of fenretinide-induced apoptosis of neuroblastoma may operate through increased intracellular levels of the lipid secondary-messen- ger ceramide (8, 13). Because fenretinide synergizes with chemother- apeutic drugs to induce apoptosis in vitro (7), defining the mechanism of apoptosis induction by fenretinide will be important in the thera- peutic application of fenretinide or in the search for other compounds that synergize with conventional chemotherapeutic drugs. Therefore, the aim of this study was to identify gene(s) induced by fenretinide that might be implicated in mediating the synergistic induction of apoptosis with chemotherapeutic reagents in neuroblastoma cells, and the mechanisms and role of ROS in these processes. We show that fenretinide, unlike 13-cis retinoic acid, or other retinoic acid isomers, induces the expression of GADD153, also known as CHOP (CEBP homology protein), a growth arrest and DNA damage-inducible tran- Received 2/13/02; accepted 8/2/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The research was funded by CLIC, Challenging Cancer and Leukemia in Childhood, The North of England Children’s Cancer Research Fund and the Wellcome Trust in the United Kingdom and by Telethon E872, Associaziore Italiana Cancro Ricerca, Ministero dell’Universita ` e Ricerca Scientifica e Tecnologica (MURST)-cofin, EU (QLG1-1999- 00739) and Consiglio Nazionale delle Ricerche in Italy. F. B. and M. R. were supported by a fellowship from Federazione Italiana Ricerca Cancro. 2 To whom requests for reprints should be addressed, at Medical Molecular Biology Group, 4th Floor, Cookson Building, Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, United Kingdom. Phone/Fax: 44-191-2228129; E-mail: chris.redfern@ncl.ac.uk. 3 The abbreviations used are: RAR, retinoic acid receptor; AA, arachidonic acid; COX, cyclooxygenase; DCFDA, dihydrodichlorofluorescein diacetate; ETI, 5,8,11-eicosa- triynoic acid; ETYA, 5,8,11,14-eicosatetraynoic acid; GSH, reduced glutathione; 12- HETE, 12 (S)-hydroxyeicosatetraenoic acid; 12-HpETE, 12 (S)-hydroperoxyeicosatetrae- noic acid; LOX, lipoxygenase; NOS, nitric oxide synthase; PI, propidium iodide; PLA 2 , phospholipase A 2 ; ROS, reactive oxygen species; Tet, tetracycline; GSSG, total glutathi- one; NAC, N-acetylcysteine; GAPDH, glyceraldehyde-3-phosphate dehyrogenase EC 1.2.1.12. 5158 Research. on November 24, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from